1531 alpn-101, a first-in-class dual icos/cd28 antagonist ... · aba+prez vs fc dpb s fc cntrl aba...
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ACR ANNUAL MEETING » NOVEMBER 8-13 » ATLANTA, GA, USA
4310
38
4
536
179
SjS SLE
PsA
3238
10
7
021
2
ALPN-101 vs Fc Aba vs Fc
Aba+Prez vs Fc
DPBS Fc Cntrl Aba ALPN-1010.0
0.5
1.0
1.5
2.0
2.5
CXCR5+ PD-1+ CD4+ Tfh cells
% o
f C
D4
+ c
ells
********
DPBS Fc Cntl Aba ALPN-1010
1
2
3
4
IFN-+ CD4+ T cells
% o
f C
D4
+ T
cells
*****
*
DPBS Fc Cntl Aba ALPN-1010
1
2
3
4
5
IL-17A+ CD4+ T cells
% o
f C
D4
+ T
ce
lls
*******
*
DPBS Fc Cntrl Aba ALPN-10140
45
50
55
60
65
70
CD19+ B cells (% of live)
% o
f liv
e c
ells
DPBS Fc Cntrl Aba ALPN-1010
2
4
6
8
10
GC cells as %B cells
% o
f C
D1
9+
B c
ells
****
DPBS Fc Cntrl Aba ALPN-1010
2000
4000
6000
Anti-Collagen Antibody
An
ti-c
ollag
en
Ig
G (
g/m
L) ****
****
Resting Restim
0
2000
4000
6000
CD28
Stim Condition
Mean
Flu
ore
scen
ce
No Skew
Th17-skew
Resting Restim
0
2000
4000
6000
8000
10000
ICOS
Stim Condition
Mean
Flu
ore
scen
ce
Th17-skew
No Skew
0
500
1000
1500
Inhibitor (3200 pM)
IL-1
7A
(p
g/m
L)
No Skewing Th17-Skewing Fc control
Prezalumab
Belatacept
Bela + WT ICOSL
Bela + Preza
ALPN-101
0
20
40
60
80
100
Inhibitior (3200 pM)
% P
roli
fera
tio
n
0
200
400
600
800
IL17A
RP
KM
FcPrezalumabAbataceptAba+PrezALPN-101PsA No Stim
0
300
600
900
1200
IL17F
RP
KM
0
300
600
900
1200
CSF2
RP
KM
0
100
200
300
CCL20
RP
KM
0
250
500
750
1000
CD28
RP
KM
0
250
500
750
1000
ICOS
RP
KM
0
500
1000
1500
2000
IFNG
RP
KM
0
500
1000
1500
2000
TNF
RP
KM
0
200
400
600
800
1000
IL2
RP
KM
0
300
600
900
IL3
RP
KM
0
200
400
600
IL13
RP
KM
0
50
100
150
IL21
RP
KM
FcPrezalumabAbataceptAba+PrezALPN-101PsA No Stim
• BACKGROUND / PURPOSE: ALPN-101 is an Fc fusion protein of a human inducible T cell costimulator ligand
(ICOSL) variant immunoglobulin domain (vIgDTM) designed to simultaneously inhibit the CD28 and ICOS
costimulatory pathways. CD28 and ICOS each play a role in T cell activation and adaptive immunity which can
contribute to autoimmune disease when dysregulated. ALPN-101 has previously been shown to have potent
immunosuppressive activity in various in vitro and in vivo models of disease, including acute graft-versus host
disease and multiple sclerosis. We report here in vitro analyses using PBMC from rheumatoid arthritis (RA) and
psoriatic arthritis (PsA) patients and from healthy donors. ALPN-101 demonstrated superior suppression of
human T cell activation and potent reduction of inflammatory mediators known to contribute to the pathogenesis
of RA, PsA, and juvenile idiopathic arthritis (JIA). Additionally, the efficacy of ALPN-101 was confirmed in vivo in
a mouse model of collagen-induced arthritis (CIA).
• METHODS: Healthy donor, RA, and PsA patient PBMC or Th17-skewed T cell cultures were stimulated with
K562 cells expressing CD80, CD86, ICOSL, and anti-CD3 (OKT3) to evaluate the potency of ALPN-101 to
suppress pro-inflammatory cytokine production. The activity of dual pathway inhibition by ALPN-101 was
compared to the CD28-only inhibitor abatacept (CTLA-4-Fc, Bristol-Myers Squibb via Catalent) and to the ICOS
pathway inhibitor prezalumab (AMG-557; anti-ICOSL, Creative Biolabs). ALPN-101 was tested in vivo against
abatacept in a CIA model in which male DBA/1 mice were immunized with bovine collagen in Freund’s adjuvant
on Days 0 and 18.
• RESULTS: Compared to abatacept, prezalumab, or combination abatacept + prezalumab, ALPN-101
demonstrated superior suppression of pro-inflammatory cytokine (i.e. TNF-α, IFN-γ, IL-2, IL-6, IL-17A, GM-CSF,
etc.) release from stimulated healthy and patient PBMCs, and suppressed T cell proliferation in Th17-skewed
cultures. The administration of ALPN-101 resulted in significant disease reduction in the mouse CIA model
(including decreased paw inflammation, serum cytokines, and anti-collagen antibodies), matching or exceeding
the activity of abatacept.
• CONCLUSIONS: The efficacy of dual CD28/ICOS antagonist ALPN-101 is superior to CD28 or ICOS
costimulatory pathway inhibitors, administered individually or in combination, in human in vitro and/or mouse in
vivo translational studies. The data suggest that ALPN-101 may significantly improve upon the clinical efficacy of
currently approved therapeutics like abatacept for treatment of inflammatory diseases, including rheumatoid,
psoriatic, and juvenile idiopathic arthritis. A Phase 1 clinical trial with ALPN-101 in healthy volunteers is ongoing
(NCT03748836), and trials in inflammatory diseases are planned.
Lawrence S. Evans, Stacey R. Dillon, Katherine E. Lewis, Susan Bort, Erika Rickel, Jing Yang, Martin F. Wolfson, Sherri Mudri, Kayla Susmilch, Steven D. Levin, Sean MacNeil, Mark W. Rixon, Jan L. Hillson, Stanford L. Peng, and Kristine M. Swiderek. Alpine Immune Sciences, Inc. Seattle, WA
ALPN-101, a First-in-Class Dual ICOS/CD28 Antagonist, Suppresses Key Effector Mechanisms Underlying
Rheumatoid and Psoriatic Arthritis
• Abstract
• ALPN-101 binds both CD28 and ICOS and demonstrates immunosuppressive efficacy superior to
CD28 or ICOS costimulatory pathway inhibitors, administered individually or in combination, in human
in vitro and/or mouse in vivo translational studies.
• The data suggest that ALPN-101 could significantly and uniquely improve
upon the clinical efficacy of currently approved therapeutics like abatacept or
IL-17 inhibitors for treatment of inflammatory diseases, including rheumatoid,
psoriatic, juvenile idiopathic and other arthritis conditions.
• A Phase 1 clinical trial with ALPN-101 in healthy volunteers is ongoing
(NCT03748836), and trials in inflammatory diseases are planned.
Summary and Conclusions
Figure 1: ALPN-101 is an ICOSL variant immunoglobulin domain (vIgDTM)
engineered for enhanced affinity for CD28 and ICOS
Figure 3: Rationale for ALPN-101 in RA, PsA or TFH Autoimmune Mediated Diseases
Sponsor: Alpine Immune Sciences, Inc. AlpineImmuneSciences.com @AlpineImmuneSci © 2019 Alpine Immune Sciences. All rights reserved.
A) ALPN-101 was titrated and incubated with CHO cells expressing human CD28 or ICOS. Bound protein was detected with anti-human IgG-PE and measured by flow cytometry.
B) ALPN-101 or comparators were titrated and incubated with mixed fixed amounts of CD86 or ICOSL labeled with AlexaFluor-647. Proteins were added to CHO cells expressing human CD28 or ICOS. After incubation, residual protein washed away and bound Alexa-647 levels measured by flow cytometric analysis. IC50 levels determined using Graphpad Prism and shown below.
Figure 4: ALPN-101 Binds CD28 and ICOS and Prevents Ligand Binding
Figure 5: Superior Inhibition of Cytokine Secretion from Stimulated Patient or
Healthy Donor PBMCs with ALPN-101
Figure 6: Significant Increase in Downregulated Genes Associated with
PsA Disease Pathogenesis by ALPN-101 Relative to Comparators
Key genes related to disease pathogenesis are highlighted in bold. More genes associated with disease pathogenesis are significantly downregulated by ALPN-101 than abatacept alone or in combination with prezalumab. ICOS-inhibition (ALPN-101 or aba plus prez) is required for down regulation of IL-17A and IL-17F. Criteria for genes reduced by ALPN-101: FDR<0.05, LOG FC<-0.5. Criteria for genes reduced by abatacept: FDR< 0.3, LOG FC <-0.5.
Significant gene reduction and/or modulation by ALPN-101 vs comparators observed for: A) Th17-associated effector molecules; B) Costimulatory molecules; C) Inflammatory TH1 or TH2-associated effector molecules; D) ALPN-101 gene signature
Figure 7: ALPN-101 Demonstrates Greater Suppression of Th17-Skewed Cells vs
CD28- or ICOS-Only Inhibitors
Primary stimulation: Human PBMC were stimulated with aAPCs and re-fed with cytokine. Cells were allowed to come to rest (~ D10 -14)
• No Skewing: K562/OKT3/CD80 + CD86 + IL-2• Th17 Skewing: K562/OKT3/ICOSL + Th17 skewing media
(Cat#CDK003, R&D Systems)
Secondary stimulation: Rested cells were re-stimulated in a mixed-lymphocyte
reaction (MLR) using up to 100,000 T-cells with 10,000 allogenic LPS-activated monocyte-derived dendritic cells with 1000 pM protein.
Assay Read-outs (4 days post-restim):• Flow cytometric analysis of ICOS, CD28 expression and T-cell
proliferation (CFSE dilution)• IL17-A secretion measured by ELISA
Figure 8: ALPN-101 is More Effective than Abatacept in a Semi-Therapeutic Dosing
Regimen in a Mouse Collagen-Induced Arthritis (CIA) Model
C) Popliteal LNs were harvested and processed into single cell suspensions, surface stained for various markers: TFH cells (CD3, CD4, CXCR5, and PD-1); germinal center (GC) B-cells (CD19, GL-7, CD95). For intracellular cytokine staining, cells were stimulated for 5 hours with PMA/Ionomycin in the presence of Golgi Stop and Golgi Plug. Cells were then surface stained for CD3, CD4, fixed, permeabilized and stained for IFN-gamma or IL-17A. D) The concentration of total anti-mouse Type II collagen IgG antibody in the sera of immunized mice was determined by ELISA (Chondrex, Cat#2036T).
Example toludine blue staining of decalcified front paws are shown:
(B) ALPN-101 Significantly Reduces Inflammation and Structural
Damage as Measured by Paw Swelling and Histological Analysis
(A) Study Design
1531
(C) Flow Cytometric Analysis of Popliteal LNs
Figure 2: ALPN-101 Blocks Both CD28 and ICOS Pathways
IgSF protein
Moderate affinity for ICOS, low affinity CD28
Random mutagenesis of ICOSL IgV domain
Parental ICOSL Selections:rCD28,rICOS
Binding assays:• Flow cytometry• Octet (affinity)
MLR
Screen yeast outputs for improved binding to ICOS and CD28
Transient 293 or CHO production followed by Protein A purification
ICOSL vIgD-Fc
Therapeutic
proteins
Bead and FACS selections
vIgD
Fc-fusion protein generation
Counter-structure binding
IgSF ECD Yeast Display Libraries
Functional assays
Sequence yeast outputs & identify unique variant hits
ALPN-101:Dual CD28 and ICOS Antagonist
Variant ICOSL lgVdomain (vlgD™)
Effectorless human lgG Fc domain(No Fc receptor binding)
Monoclonal antibody~150 kDa
ALPN-10180.8 kDa
lgG Fcdomain
• Increased affinity for CD28• Increased affinity for ICOS
• ALPN-101 inhibits both CD28 and ICOS-mediated costimulation. Comparators such as abatacept /
belatacept (CTLA4-Ig, BMS) or FR104 (anti-CD28 Fab, OSE Immunotherapeutics) inhibit only the CD28/CTLA-4 pathway, while prezalumab (anti-ICOSL mAb, Amgen) inhibits only ICOS costimulation.
• CD28 costimulation is critical for naïve and memory CD4+ T cell activation. ICOS signaling augments CD28 costimulation but also plays non-redundant roles; ICOS deficiency leads to defective humoral responses in both mice and humans (Panneton2019 Imm Revs).
• ICOS signaling is crucial for the development of human TH17 cells (Paulos 2010 Sci Transl Med) and function and maintenance of follicular helper T-cells (TFH, Weber 2015 JEM). ALPN-101 may thus be uniquely suited to treat diseases driven by these pathogenic cell types.
Ligand Binding IC50s (nM)
Male DBA/1 mice were immunized with bovine collagen in complete Freund’s adjuvant (CFA) on Day 0 and boosted with bovine collagen/incomplete Freund’s adjuvant (IFA) on Day 18 (Hooke Laboratories, Lawrence, MA)
Mice were treated intraperitoneally (IP) every 3 days for a total of 5 doses (Q3Dx5) with vehicle (Dulbecco’s phosphate buffered saline/DPBS), Fc control, abatacept (Orencia™), or ALPN-101 in a semi-therapeutic regimen, starting before administration of the collagen boost on Day 18.
0 1 2 3 4 5 6 WeeksInject bovine collagen in
CFA (tail)
Boost: Inject bovine
collagen in IFA (tail)
Study end
Dosing
IL-6 IL-13 IL-17A TNF-
0
200
400
600
0
25
50
75
Serum Cytokine Levels in DPBS Treated Mice
IL-6
(p
g/m
L)
IL-1
3, IL
-17A
, TN
F-
(pg
/mL
)
E) Average serum cytokine levels were determined for DPBS-treated mice. % Inhibition of cytokine was determined for individual mice from the Fc control, Abatacept, or ALPN-101-treated mice relative to DPBS
mice using the following formula: ((Ave DPBS – Exp Animal)/Ave DPBS))*100
TX Cytokine
GM-CSF -195 -100 -88 53 -7 -52 28 46 48 46 35 39 62 51 16 52 55 30 40 7 26 31 31 45 52 39 38 41 3 40 68 69 37 49 50 65 49 19
IFNG -218 -51 -409 -153 -143 -8 -158 -20 -7 -7 9 -29 -11 -45 -101 -25 -11 -28 -5 -1 5 -5 -11 -2 -39 20 26 19 -143 -12 9 -11 -4 4 -1 -10 -7 3
IL-12 p70 -6700 -234 -245 -87 -126 -302 -420 -68 -191 -75 -9 -109 -78 13 -204 26 -53 -157 1 7 -3 -60 25 -2 -49 43 43 3 -128 -73 -135 -84 -15 -72 37 35 39 34
IL-13 -93 -42 6 48 47 40 52 56 57 44 44 53 59 52 47 41 56 48 53 48 42 55 56 48 54 48 52 51 22 41 57 62 56 62 53 44 61 63
IL-17A -99 -138 -58 45 -24 14 59 41 55 63 39 64 59 44 43 50 70 70 57 43 53 57 70 58 63 54 73 49 21 47 60 56 63 46 61 55 55 39
IL-17F -1351 -1290 -339 -24 -416 -177 -8 -79 -3 24 -36 -38 -34 -18 -87 -4 13 11 -25 -29 -2 -11 41 15 -19 18 42 -10 -160 13 18 21 16 6 36 21 6 -13
IL-2 -2831 -337 -1188 -3788 -572 8 0 0 -13 0 -17 0 0 -2 2 -6 -24 -10 15 3 -8 -5 -11 15 0 -11 -5 0 -744 -4 -58 -27 -4 -3 -22 33 -36 -21
IL-21 -3632 -869 -408 -75 -283 -172 -174 -5 -10 13 32 -32 -24 16 -45 24 21 -8 -13 7 17 5 32 6 23 22 53 30 -600 11 8 52 3 11 22 62 49 32
IL-6 -108 -285 -723 -104 -110 -279 -437 -721 -69 -138 -90 -217 -85 -12310 -703 -529 -381 -63 -154 0 -1 -87 -8 -146 -15 -184 -283 -19 -147 -27 -128 -26 -15 -117 -100 -128 -42 -5
TNF-alpha -160 -253 -21 2 -57 -33 -3 8 8 16 -4 1 9 26 -3 -13 5 1 0 -13 0 2 19 0 5 30 20 24 -112 -4 31 0 16 17 12 38 18 24
GM-CSF -103 -31 -6 70 20 23 73 67 70 61 41 61 84 74 39 64 57 45 51 43 57 39 35 63 62 55 46 60 17 68 72 78 53 49 67 75 75 68
IFNG -58 18 -75 -14 18 22 49 61 24 42 48 61 55 40 46 37 63 57 65 17 54 68 29 64 63 73 64 68 -36 -4 36 20 22 36 47 34 23 27
IL-12 p70 -2973 -45 -49 5 26 0 2 62 7 24 46 50 36 13 33 79 87 20 56 89 68 46 68 86 78 78 82 82 -69 61 1 28 64 75 85 74 92 78
IL-13 -97 -15 7 33 33 23 57 42 62 24 47 49 62 18 44 36 55 36 60 70 54 56 70 50 66 47 45 46 25 57 58 70 66 64 60 56 75 74
IL-17A -54 -152 -28 41 -5 -3 59 50 45 30 31 60 56 17 12 37 9 43 28 13 15 28 48 6 34 28 56 8 6 24 36 48 28 19 35 48 59 34
IL-17F -98 -216 -59 47 -14 -22 57 57 51 34 43 70 59 28 26 45 28 45 38 5 28 52 44 50 48 49 69 21 -25 11 33 55 31 27 43 42 50 41
IL-2 -43 51 -4 -4 56 92 98 86 64 71 43 84 93 92 91 79 72 54 84 58 64 90 24 89 93 90 92 94 53 88 75 85 51 61 45 90 39 55
IL-21 -1195 -161 -23 88 47 26 84 83 87 63 85 89 85 44 41 75 84 86 73 81 82 83 87 83 74 83 89 82 -11 88 86 91 79 91 89 93 96 94
IL-6 -13 -26 -17 44 17 24 48 62 55 30 59 55 63 100 42 41 57 33 41 84 37 59 62 48 63 56 22 65 12 49 42 59 50 55 55 43 60 66
TNF-alpha -21 -23 2 42 48 13 15 15 7 18 -1 22 27 69 23 10 12 3 9 9 28 56 31 43 9 73 41 49 33 22 37 40 51 54 35 64 21 63
GM-CSF 28 -5 52 87 63 53 83 85 89 86 82 84 94 97 87 90 92 79 94 85 86 93 78 95 94 90 88 92 60 82 88 88 82 90 91 85 85 89
IFNG 4 32 8 -8 43 47 79 78 61 90 73 77 75 73 89 66 90 92 92 77 79 98 81 93 85 95 85 90 49 12 65 77 58 78 84 53 29 70
IL-12 p70 -1143 -52 -20 66 13 26 44 80 83 90 77 78 80 77 51 82 94 63 75 97 68 82 80 91 92 91 84 85 63 84 88 56 77 88 93 69 93 79
IL-13 38 6 46 70 76 58 75 80 87 77 86 75 89 95 88 86 92 73 95 96 89 98 87 95 96 93 92 92 58 79 86 88 93 96 91 76 89 97
IL-17A 31 -7 32 77 43 55 78 84 82 80 81 90 89 91 91 90 93 95 96 90 92 95 93 95 97 93 97 93 53 76 85 73 93 86 89 77 81 93
IL-17F 46 -19 14 83 38 35 71 90 82 85 88 94 90 95 88 88 89 97 97 84 85 97 89 98 93 89 99 92 51 59 89 74 89 88 85 70 75 96
IL-2 27 63 3 42 72 95 100 96 97 98 87 92 100 100 100 100 100 92 100 100 99 100 96 100 100 100 100 100 67 98 86 98 100 100 100 97 100 100
IL-21 -300 -115 -158 95 -20 46 84 98 97 87 95 97 94 89 61 87 98 97 95 98 96 99 97 99 98 97 97 98 66 95 97 94 94 97 97 96 96 97
IL-6 34 14 16 72 41 52 100 77 79 81 85 68 93 100 43 60 92 75 95 97 76 100 89 98 97 95 99 100 48 65 81 41 90 97 82 15 67 93
TNF-alpha 8 -13 6 83 78 16 21 25 20 36 18 39 40 96 37 46 23 15 18 76 87 96 81 93 14 95 85 72 63 50 88 67 88 91 80 78 48 92
Pre
zalu
ma
bA
ba
tac
ep
tA
LPN
-10
1
RA (N =13) PsA (N =15) HD (N=10)
% Inhibition(Relative to Fc
Control)
>0
50
100
TX Cytokine
IL-6 0 7 21 11 39 44 47 70 51 49 47 52 42 66 27
IL-13 0 4 0 24 4 0 0 0 24 14 43 43 0 43 4
IL-17A 0 0 0 0 0 0 0 0 11 0 4 0 0 6 73
TNF-a 0 0 0 0 0 2 0 0 0 30 0 0 63 0 59
IL-6 - 56 67 41 75 57 58 84 69 99 34 46 74 74 97
IL-13 - 4 14 24 24 43 43 34 43 43 62 24 34 24 43
IL-17A - 0 0 0 0 0 0 0 0 0 3 99 0 73 99
TNF-a - 0 0 23 0 0 0 0 7 0 55 0 69 5 0
IL-6 71 91 99 78 92 99 94 99 93 99 99 100 100 100 100
IL-13 34 48 43 34 53 70 34 53 53 53 53 87 87 87 87
IL-17A 0 0 0 0 11 0 1 67 99 23 99 99 99 99 99
TNF-a 0 0 0 40 28 23 72 11 0 82 28 99 99 99 99
Fc
Co
ntr
ol
Ab
ata
ce
pt
ALP
N-1
01
Serum Cytokine Inhbition
% Inhibition
(Relative to DPBS)
>0
50
100
• FAM131C
• GRB7
• CABLES1
• XIRP1
• FST
• CDO1
• TAC1
• ALCAM
• G0S2
• TMCC2
• IL1R1
• KCNG1
• DIRAS3
• IGSF3
• CALD1
• IL12B
• IL2
• IFNG
• IL21• IL3
• KIF3B
• BATF3
• CCL17
• CTLA4
• CXCR5
• FOSL1
• GATA3
• ICOS
• ID2
• IL12RB2
• IL17F
• IL18RAP
• IL1R2
• IL1RL2
• IL23R
• IL6• LIF
• LTA
• OSM
• TNF• VEGFA
• BCL2L1
• CCL20
• CD40LG
• CSF2
• IER3
• IL13
• IL17A• IL24
• IL31• IL4
• IL5
• IL9
A) Cytokine secreted from RA, PsA, or healthy donor stimulated PBMCs. B) % Inhibition determined using the following formula: ((Fc control value – Exp value)/Fc control value))*100. For most analytes, ALPN-101 demonstrated greater cytokine inhibition than observed with abatacept or prezalumab alone or combined (i.e. IL-17A).
A) Target Binding B) Blockade of Ligand Binding
to Target Receptor
C) Costimulation Blockade
A) TH17-Associated Effector Molecules
C) Inflammatory TH1 or TH2-Associated Effector Molecules
Cytokine Secretion
CD4+ T-cell Proliferation
(E) Greater Inhibition of Cytokine in Sera of Mice Treated with ALPN-101
The study was terminated on Day 35. Endpoints include: Disease scores; flow cytometric analysis of popliteal lymph nodes; serum cytokine analysis; anti-collagen IgG analysis
Inflammation Pannus formation Cartilage damage Bone resorption
0
5
10
15
20
25
Paw Histology Scores, by Parameter (Mean+SD)
ParameterAv
era
ge s
co
re p
er
mo
use
(m
ean
+S
D)
DPBS
Fc Control
Abatacept
ALPN-101
• CCL20
• CD40LG• CSF2
• ICOS
• ID2
• IFNG• IGSF3
• IL17A• IL1R1
• IL2
• IL21• IL24
• IL3
• IL31
• IL6• IL9
• LIF
• NFKBID
• PDCD1
• TNF
• BATF3
• CTLA4
• FLT1
• FOSL1
• IL13
• IL17F• IL1R2
• IL23R• IL4
• IL5
• VEGFA
• CCL17
• CXCR5• GATA3
• IL12RB2
• IL18R1
• IL18RAP
• KITLG
• LTA
• PVR
• SOCS3
• TFRC
• TNFRSF8
D) ALPN-101 Gene Signature
10 100 1000 10000 1000001000000
0
10000
20000
30000
40000
50000
CD28
[ pM ]
Med
ian
Flu
ore
scen
ce
WT ICOSL
ALPN-101
Abatacept
Belatacept
Prezalumab
100 1000 10000 100000 1000000500
1000
1500
2000
CD86 CD28
[ pM ]
Med
ian
Flu
ore
scen
ce
ALPN-101
Abatacept
Belatacept
10 100 1000 10000 1000001000000
0
20000
40000
60000
ICOS
[ pM ]
Med
ian
Flu
ore
scen
ce
WT ICOSL
ALPN-101
Abatacept
Belatacept
Prezalumab
100 1000 10000 100000 10000000
25000
50000
75000
ICOSL ICOS
[ pM ]
Med
ian
Flu
ore
scen
ce
WT ICOSL
Prezalumab
ALPN-101
1 100 10000 1000000700
1050
1400
1750
2100
ICOS Costimulation Blockade
[ pM ]
RL
U
Prezalumab
ALPN-101
1 100 10000 10000000
3000
6000
9000
CD28 Costimulation Blockade
[ pM ]
RL
U
Belatacept
ALPN-101
Abatacept
B) Costimulatory Molecules
C) CD28 costimulation blockade demonstrated by inhibition of artificial APC (aAPC) expressing OKT3 + human CD86 from stimulating via endogenous CD28 on Jurkat/IL-2 cells expressing luciferase driven from an IL-2 promoter (Promega). ICOS blockade demonstrated by inhibition of aAPC with OKT3 + human ICOSL from stimulating ICOS on Jurkat/IL-2 cells transduced with extracellular domain of human ICOS and intracellular domain of CD28.
Patient or healthy donor peripheral blood mononuclear cells (PBMC) were stimulated with fixed artificial APCs (shown at right) for 48 hours in the presence of ALPN-101 (100nM) or comparators:
• Prezalumab (AMG-557, anti-ICOSL, Creative BioLabs)• Abatacept (CTLA4-Ig)• Abatacept + prezalumab
Assay Read-outs:• Cytokine secretion measured by ELISA or multi-plex
analysis (EMD Millipore)• RNAseq analysis
A) Cytokine Secretion from Stimulated PBMC B) Percent Inhibition of Cytokine Secretion Relative to Fc Control
Genes significantly down-regulated in response to ALPN-101 in PsA, SjS, and SLE stimulated patient populations suggest ALPN-101 can impact pathogenic genes and/or pathways implicated across many autoimmune diseases.
• A) Severe diffuse inflammatory infiltrate with severe edema with total or near total destruction of joint architecture, with multifocal chrondrocyte loss and full thickness defects in cortical bone with destruction of joint architecture in a PBS-treated animal.
• B) Severe diffuse inflammatory infiltrate with severe edema with extensive destruction of joint architecture, with marked chondrocyte loss and full thickness defects in cortical bone with distortion of the cortical surface in a Fc-treated animal.
• C) Moderate to severe inflammatory infiltrate with marked destruction of joint architecture, minimal to mild disruption of structure and some areas of bone resorption in an abatacept-treated animal.
• D) Absence of inflammation, pannus, cartilage damage or bone resorption in an ALPN-101-treated animal. Bar, 500 µm.
Please see also: Dillon et. al. ALPN-101, a First-in-Class Dual ICOS/CD28 Antagonist, Suppresses Key Effector Mechanisms Associated with Sjögren’s Syndrome
and Systemic Lupus Erythematosus (Poster #2416; Sjögrenʼs Syndrome – Basic & Clinical Science Poster I, Tuesday, November 12, 2019, 9-11 AM)
Artificial APCK562/OKT3/CD80/CD86/ICOSL
T-cell
CD28
ICOS
CD80/86
ICOSL
OKT ScFv
Target StructureAntagonists
ICOSWT ICOSL IgV
ICOSLPrezalumab
CD80/ CD86
Abatacept
Belatacept
ALPN-101CD28
ICOS
CD80/86(B7)
CD28
T cell
prezalumab
ICOSL
ICOS
T cellactivation
APC/B cell
CD80/86(B7)
CD28
CTLA4-lg
T cellactivation
ICOSL
ICOS
ICOSL
ICOS
CD80/86(B7)
CD28
T cell Inactivation
ALPN-101
Paw Scores Over Time (P values)
(D) Anti-Collagen Ab Response
0
500
1000
1500
2000
2500 IL-17A Response
IL-1
7A
(p
g/m
L)
0
40000
80000
120000 IFN- Response
IFN
- (
pg
/mL
)
0
50
100
150
200
250 TNF- Response
Donor Type
TN
F-
(n
g/m
L)
RA PsA HD
γ
A. PBS B. Fc
C. Abatacept D. ALPN-101
15 20 25 30 350
2
4
6
8
10
12
14
16
Paw Scores Over Time (MeanSEM)
Day After Immunization
Pa
w S
co
re (
ma
x =
16) DPBS
Fc Control
Abatacept
ALPN-101
±Paw Scores Over Time (Mean±SEM)
Cell Surface
Expressed
Target
CD28 ICOS
Ligand CD80 CD86 ICOSL
Inh
ibito
r
WT ICOSL - - 17.2
Prezalumab - - 23.3
Abatacept 5.4 12.9 -
Belatacept 2.5 2.8 -
ALPN-101 1.0 0.7 4.3
IndicationPathogenic
Cell Type
Clinical Effect of
CD28 Inhibition
ALPN-101 In Vivo
Model EfficacyPotential Role for ICOS in Disease Pathogenesis
RA T & B-cellsAbatacept approved (2005)
Yes↑ PD-1+ ICOS+CXCR5- CD4+ T cell population in joints and blood that provide B-cell help and correlate with seropositivity
(rheumatoid factor or anti-citrullinated peptide) in RA patients (Rao 2017 Nature)
PsA TH17Abatacept approved (2017)
YesICOS – ICOSL critical for the development of TH17 cells, increased ICOS expression on TH17 vs TH1 or TH2 cells (Paulos 2010
Sci Transl Med)
Sjogren’s (SjS) T & B-cells
Abatacept failed to meet primary endpoints (NCT02915159)
Yes↑ numbers of circulating ICOS+ TFH cells correlating with ↑ plasmablasts, plasma cells, and autoantibody levels (Brokstad
2018 Scand J Immunol); ↑ numbers of circulating ICOS+ TFH cells correlates with anti-SSA/Ro 60, anti-SSA/Ro 50 ab levels
and ESSDAI scores (Fonseca 2018 Arthritis Rheumatol)
Systemic Lupus
Erythematosus
(SLE)
T & B-cellsAbatacept failed to prevent disease flare (NCT00119678)
Yes↑ numbers of ICOS+ TFH cells correlating with ↑ circulating plasmablasts, levels of serum anti-dsDNA and anti-nuclear
antibodies (ANA)(Zhang 2015 Lupus); ↑ ICOS+ TFH cells correlate with SLEDAI, circulating plasmablasts, and anti-dsDNA
positivity, reflecting active disease (Choi 2015 Arthritis Rheumatol)
DPBS Fc Control Abatacept ALPN-101
DPBS - - 0.0006 <0.0001
Fc Control - - 0.0172 <0.0001
Abatacept - - - 0.0016
ALPN-101 - - - -
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